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Low Blood CD8+ T-Lymphocytes and High Circulating Monocytes Are Predictors of HIV-1-Associated Progressive Encephalopathy in Children

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Low Blood CD8

T-Lymphocytes and High Circulating Monocytes Are

Predictors of HIV-1-Associated Progressive Encephalopathy in Children

Silvia Sa´nchez-Ramo´n, MD, PhD*; Jose´ MaBello´n, MsC*; Salvador Resino, PhD*; Carmen Canto´-Nogue´s, PhD*; Dolores Gurbindo, MD‡; Jose´-Toma´s Ramos, MD§; and

MaAngeles Mun˜oz-Ferna´ndez, PhD*

ABSTRACT. Objective. Human immunodeficiency virus type 1 (HIV-1)-associated progressive encephalop-athy (PE) is a common and devastating complication of HIV-1 infection in children, whose risk factors have not yet been clearly defined. Regardless of the age of presen-tation, PE shortens life expectancy. Paradoxically, as sur-vival of patients has been prolonged as a result of the use of antiretroviral therapy, the prevalence of PE has in-creased. Therefore, a predictive marker of PE emergence is critical. The objective of this study was to determine in an observational study whether any immunologic (CD4

and CD8T-lymphocyte counts, monocyte counts) or

virologic (viral load [VL], biological characteristics of viral isolates) marker might be predictive of PE and whether any particular marker may be involved in the timing of clinical onset of PE.

Methods. A total of 189 children who were vertically infected with HIV-1 were studied retrospectively, 58 of whom fulfilled criteria of the American Academy of Neurology for PE. T-lymphocyte subsets and monocytes in peripheral blood were quantified by flow cytometry. HIV-1 RNA was measured in plasma using a quantitative reverse transcriptase polymerase chain reaction assay. Demographic, clinical, and viro-immunologic character-istics in infants were compared with control groups us-ing logistic regression. Proportions were compared usus-ing the2 test or Fisher exact test. For each child,

immuno-logic and viroimmuno-logic markers were analyzed in parallel closely before clinical onset of PE and closely after PE onset and compared by using the Studentttest for paired samples.

Results. Overall, mortality of 58 HIV-1-infected chil-dren who developed PE was significantly higher than of children who did not develop this complication. Blood CD8T-lymphocytes <25% in the first months of life

suggested a relative risk of progressing to PE 4-fold higher than those with CD8>25% (95% confidence

interval: 1.2–13.9) and remained statistically significant after adjustment for treatment. When we compared the PE-positive group with the acquired immunodeficiency syndrome (AIDS)/PE-negative group (children who de-veloped clinical category C and without neurologic man-ifestations) in a cross-sectional study within 12 months before PE or AIDS diagnosis, respectively, the %CD8

T-lymphocytes were significantly lower in the

PE-posi-tive group. Normalized absolute counts of CD8

T-lym-phocytes with respect to seroreverting children were sig-nificantly lower in the group of children with encephalopathy with respect to the AIDS/PE-negative group (data not shown). It is interesting that a statisti-cally significant increase was observed in circulating monocyte percentages and absolute counts shortly before the first neurologic symptoms compared with values af-ter PE was established and with those from HIV-1-in-fected controls. With respect to AIDS-related events, PE was strongly associated with anemia and lymphoid in-terstitial pneumonitis in the PE-positive group with re-spect to a group of children with AIDS but without PE.

Conclusion. HIV-1 infection of the central nervous system (CNS) remains an important clinical concern. The first step toward PE prevention in HIV-1-infected chil-dren should be directed at predicting risk of PE and thus the prompt and reliable identification of infants who are at risk for CNS disease progression. Low blood CD8

T-lymphocytes is a strong early predictive marker of PE emergence in vertical HIV-1 infection. Indeed, among all of the immunologic and virologic variables assessed in this observational study, the only significant difference during the first months of life are the CD8

T-lympho-cytes. A peak of significantly higher peripheral mono-cytes before the onset of PE with respect to established PE has not been previously described, and strengthens the growing evidence that an increased traffic of mono-cytes to the brain may be a key factor in triggering neurologic symptoms. The suppression of HIV-1 replica-tion is dependent on the presence of a relatively small number of HIV-1-specific CD8T-lymphocytes, and it is

possible that the duration of the neurologically asymp-tomatic phase for any given child may depend mostly on the magnitude of specific CD8T-lymphocyte responses.

Thus, a decrease of CD8T-lymphocytes would

dimin-ish the host capacity to control viral infection, as reported in animal models, enabling infected macrophages to cross the blood-brain barrier. Our results advocate the use of CD8T-lymphocyte and monocyte counts to

fol-low-up HIV-1-infected children. We suggest that CD8

T-lymphocytes may be the nexus for many different as-pects of the disease, namely loss of control of HIV-1 replication determining higher VL, increased traffic of activated and/or infected monocytes, spread of infection to immune sanctuaries, and finally clinical neurologic emergence of PE. Moreover, we suggest that CD8

T-lymphocytes or/and monocytes may be used as putative biological markers of neuropathogenicity. This might suggest their use in decision making of when to start more effective antiretroviral regimens for HIV-1 infec-tion of the CNS and the need of new therapies either to preserve or to augment an adequate CD8T-lymphocyte

immune response. Early detection of children who are at From the Units of *Neuroimmunology and ‡Immunopediatrics, Hospital

General Universitario Gregorio Maran˜o´n, Madrid, Spain; and §Department of Pediatrics, Hospital Doce de Octubre, Madrid, Spain.

Received for publication Jun 24, 2002; accepted Sep 19, 2002.

Reprint requests to (M.A.M.-F.) Unidad de Neuroinmunologı´a, Hospital General Universitario “Gregorio Maran˜o´n,” c./Doctor Esquerdo 46, 28007, Madrid, Spain. E-mail: mmunoz@cbm.uam.es

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risk for developing PE is particularly important because aggressive highly active antiretroviral therapy improves neurologic symptoms, allows possible use of neuropro-tective treatment to prevent further development of en-cephalopathy, and emphasizes the relevance of develop-ing therapies aimed to enhance CD8T-lymphocyte

function. In conclusion, the surrogate markers routinely used in clinical practice for HIV-1 infection (ie, CD4

T-lymphocyte counts and VL) seem to be insufficient to evaluate the clinical involvement of the CNS. Other sys-temic markers, as the recent proposed markers for PE evolution (cerebrospinal fluid VL by lumbar puncture and brain atrophy by cerebral magnetic resonance imag-ing) are undoubtedly more invasive than measuring CD8T-lymphocyte and monocyte counts, when the

neurologic manifestations of PE are still preventable.

Pediatrics 2003;111:e168 –e175. URL: http://www. pediatrics.org/cgi/content/full/111/2/e168;central nervous system, HIV-1-related encephalopathy, development, pedi-atric AIDS, viral load.

ABBREVIATIONS. HIV-1, human immunodeficiency virus type 1; PE, HIV-1-associated progressive encephalopathy; AIDS, acquired immunodeficiency syndrome; ART, antiretroviral therapy; VL, viral load; CNS, central nervous system; CT, computed tomogra-phy; SI, syncytium-inducing; CTL, cytotoxic T-lymphocyte; LIP, lymphoid interstitial pneumonitis.

H

uman immunodeficiency virus type 1

(HIV-1) displays tropism for both the im-mune and the nervous systems. HIV-1-asso-ciated progressive encephalopathy (PE), among other acquired immunodeficiency syndrome (AIDS)-defining illnesses, has been considered much more common and severe in pediatric patients than in adults.1,2 It was hypothesized recently that neuro-logic symptoms that occur within the first year of life may have different pathophysiologic mechanisms and clinical significance than those that occur later, which may in turn reflect the specific timing (in utero vs perinatal) of HIV-1 infection.1 Regardless of the age of presentation, PE shortens life expectancy; es-timates of median survival after the onset of PE have ranged from 6 to 22 months.3–5

Antiretroviral therapy (ART) has forever changed the landscape of HIV-1 infection in children,6 – 8 re-ducing opportunistic infections and enabling im-mune reconstitution.9 –11 However, the impact of new ART regimens on HIV-1 infection of the devel-oping brain is still unknown.12 Some patients de-velop PE despite multidrug therapies, and others develop subtle neurobehavioral changes that dimin-ish the quality of their lives, despite the increased survival time.13Likewise, as survival of patients has been prolonged, the prevalence of PE has in-creased.14

Therefore, a predictive marker of PE emergence is critical. In this regard, preliminary results in a pro-spective study showed that peripheral blood CD8⫹ T-lymphocytes in the first months of life of HIV-infected infants were predictive of emergence of neu-rologic symptoms related to the PE clinical picture. More important, CD8⫹ T-lymphocytes were a pre-dictive marker for PE independent of the CD4⫹

T-lymphocyte and viral load (VL).15Here, we sought to

determine in an observational study whether any immunologic (CD4⫹ and CD8⫹ T-lymphocyte counts, monocyte counts) or virologic (VL, biological characteristics of viral isolates) marker might be pre-dictive of PE and whether any particular marker may be involved in the timing of clinical onset of PE. To evaluate this hypothesis, we initially examined the occurrence of PE in a cohort of vertically HIV-1-infected children. Next, we tried to identify risk fac-tors both in the mother and in the infant that can predict the risk of developing PE. We subsequently evaluated all of those parameters of children whose encephalopathy had an early onset and compared them with those of children with later-occurring PE.

METHODS Study Subjects

Between December 1984 and November 2000, 189 children who were born to HIV-1-infected mothers were diagnosed with vertical HIV-1 infection as previously described16and were followed from birth at the General University Hospital Gregorio Maran˜o´n and Doce de Octubre Hospital in Madrid, Spain. HIV-1-infected in-fants were classified according to the Centers for Disease Control and Prevention guidelines.17Fifty-eight (PE-positive group) of the 189 children met criteria of PE according to the American Acad-emy of Neurology criteria.18Encephalopathy was defined as prob-able when the following criteria were fulfilled: evidence of sys-temic infection by HIV-1; at least 1 of the following progressive findings: failure to attain or the loss of developmental milestones or of intellectual ability, impaired brain growth or an acquired microcephaly and an acquired symmetric motor deficit verified by clinical examination (eg, slowed rapid movements, abnormal gait, limb incoordination, hyperreflexia, hypertonia, or weakness), neu-ropsychological test (eg, fine motor speed, manual dexterity, per-ceptual motor skills), or both; and another cause, including active central nervous system (CNS) opportunistic infection or malig-nancy, psychiatric disorders (eg, depressive disorder), alcohol or substance use, or acute or chronic substance withdrawal, must be ruled out. Taking into account that cumulative incidence of PE from birth in the 189 HIV-1-infected children was maximal at 24 months of age (19.6%), representing 64% of the 58 children with PE, we studied thereafter separately children who developed PE before and after this age point, as “early-onset PE” and “late-onset PE” groups. PE diagnosis was made by the same attending pedi-atrician and neuropedipedi-atrician at each center. Each of the 58 chil-dren of the PE-positive group had an extensive medical history, general physical examination, serial head circumference measure-ments, and detailed neurologic examination. Two additional groups of 28 children (the PE-negative group) with vertical HIV-infection in all clinical categories—A (n⫽14), B (n⫽2), and C (n⫽12)—and 32 children who developed category C (the AIDS/ PE-negative group) but without neurologic manifestations, were randomly selected for comparisons at birth and matched to those in the PE-positive group with respect to age at AIDS presentation, respectively. Control groups had similar demographic character-istics and mothers with similar risk behaviors and were born in the same time points.

Gestational age was determined by a combination of prenatal ultrasonography, physical examination of uterine fundal height, and menstrual history. Prenatal drug exposure was defined by maternal prenatal use of illicit drugs (opiates, cocaine, or other injectables) or methadone, as assessed by either a positive inter-view and/or urine toxicological studies. Patients were considered positive when either criteria was positive. Weight and heightz scores and head circumferences percentiles were determined by using the Orbegozo’s scale for Spanish children as standards.

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Virus Isolation and Determination of Viral Phenotype

HIV-1 was isolated by a microculture technique, as previously described.16,19 Briefly, peripheral blood mononuclear cells were isolated from blood by Ficoll-Hypaque density gradient centrifu-gation (Pharmacia, Uppsala, Sweden). Both viral culture and poly-merase chain reaction assays were performed on the same sample. The criteria for a positive co-culture and determination of the days to first positive were based on p24 HIV results obtained from biweekly culture samples. According to the lag phase before p24 antigen detection and to the level of p24 antigen production by peripheral blood mononuclear cells, HIV-1 isolates from these patients were classified as rapid/high or slow/low.20We consid-ered an isolate as syncytium-inducing (SI) when both formation of syncytia in MT-2 cell line under light microscopy and p24 antigen production in the culture supernatants were detected.16Moreover, as MT-2 cell line expresses CD4 receptor and CXCR4 (X4) co-receptor but not CCR5 (R5), this cell line may be useful to differ-entiate X4-using or SI from R5-using or non-SI strains. HIVBAL(R5 or non-SI) and HIVNL4.3(X4 or SI) are routinely used as controls.

Quantitative HIV-1 RNA Assay

Plasma samples were collected into ethylenediaminetetraacetic acid tubes and separated within 4 hours, and the plasma was stored at⫺70°C. HIV-1 RNA was measured in 200␮L of plasma using a quantitative reverse transcriptase polymerase chain reac-tion assay (Amplicor Monitor, Roche Diagnostic System, Brand-ersburg, NJ).

Quantification of T-Cell Subsets and Monocytes in Peripheral Blood

T-lymphocyte subsets and monocytes in peripheral blood were quantified by direct immunofluorescence using monoclonal anti-bodies of the T series and for monocytes (anti-CD14) and flow cytometry (FACScan; Becton-Dickinson, San Jose CA), as previ-ously described.16,19

Statistical Methods

In all analyses, HIV-1 RNA levels were transformed to log10 scale to normalize their distribution. Proportions were compared using the␹2test or Fisher exact test for expected values below 5. Quantitative variables were expressed as means⫾standard error of the mean and compared by using the Student t test or the nonparametric Wilcoxon rank sum test. Kaplan-Meier estimates of the cumulative probability were used for each covariate on the risk of reaching an outcome event and were compared by the log rank test (Mantel-Haenzel). The time to progress to PE and its relative risk were estimated by the proportional hazard Cox

re-gression equation. For ruling out the effect of age on the %CD4⫹

and %CD8⫹T-lymphocytes, their values were adjusted with

re-spect to a cohort of age-matched HIV-1-exposed but uninfected children or seroreverting children (zscore).

RESULTS

PE incidence dramatically fell since 1997 from 9.5% to 5.6% in our series, in parallel with the de-creased incidence of vertical HIV infection. From 1998, both (vertical HIV infection and PE) incidences are near 0%, as a result of ART during pregnancy and early in life.

Overall, mortality of 58 HIV-1-infected children who developed PE was significantly higher than that of children who did not develop this complication (131 children: 60% vs 44% among non-PE children;

P⫽.04).

Characteristics at Birth

Sociodemographic, maternal, and birth character-istics of infants were similar for positive and PE-negative groups (Table 1). No significant differences were noted at birth between early- and late-onset PE-positive and PE-negative groups, respectively (data not shown).

Time to progress to PE was assessed by Kaplan-Meier estimates for the variables studied. Children with low birth weight (⬍2500 g) progressed to PE faster than those with birth weight⬎2500 g (P⫽.01; Fig 1A). By Cox regression analysis, children with low birth weight were 2.2-fold more likely to progress to PE than children with birth weight ⬎2500 g (95% confidence interval: 1.1– 4.1;P ⬍.01). None of the other variables examined was signifi-cantly related to risk for progressing to PE.

Risk Factors of PE in the First Months of Life

We then analyzed the predictive power of immu-nologic (%CD4⫹ and %CD8⫹ T-lymphocytes) and virologic (VL) markers in the first months of life

TABLE 1. Clinical and Immunologic Characteristics of the PE-Negative and the PE-Positive Groups at Birth and at a Time Point Within the First Months of Life (Range: 2–7 Months)

Characteristic PE-Negative Group

(n⫽28)

PE-Positive Group (n⫽58)

At birth

Gender (M/F) 7/21 23/35

Gestational age at delivery⬍37 wk 4 (20%) 13 (32%)

Drug exposure during gestation (n[%]) 6 (21.4%) 15 (26%)

Birth weight

Mean⫾sem (g) 2.656⫾132 2.708⫾82

Low birth weight (⬍2500 g) 8 (34.8%) 19 (35.8%)

Responsible for the child’s care

Biological mother 11 (58%) 35 (67%)

Other 8 (42%) 17 (33%)

In first months of life

Age (mo) 5.0⫾0.3 4.4⫾0.3

CD4⫹T-lymphocytes 36.62.8 34.43.3

CD8⫹T-lymphocytes 26.32.5 20.22.4*

VL (log10) 4.7⫾0.3 5.1⫾0.2

CD4⫹25% T-lymphocytes 24% 21%

CD8⫹25% T-lymphocytes 40.7% 85%†

VL⬎5 log10 44% 50%

Values are expressed as mean⫾SEM. SEM indicates standard error of the mean. *P⬍.05.

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(range: 2–7 months) for PE emergence (Table 1). The %CD8⫹ T-lymphocytes was significantly lower in the PE-positive group (P⬍.05) and in children with early-onset PE (P ⬍ .05) than in the PE-negative group. In addition, we studied time of progression to PE by Kaplan-Meier estimates according to a cutoff level for each of the 3 markers, 25% CD4⫹, 25% CD8⫹, and 5 log10VL, which have been reported to have prognostic value.21Children with CD8

T-lym-phocytes⬍25% in the first months of life progressed earlier to PE than those with CD8⫹ T-lymphocytes ⬎25% (P ⫽ .02), being the median time of progres-sion to PE in the former of 16.7 months versus 55.0 months, respectively (Fig 1B). By Cox regression analysis, CD8⫹ T-lymphocytes ⬍25% in the first months of life suggested a relative risk of progress-ing to PE 4-fold higher than those with CD8⫹⬎25% (95% confidence interval: 1.2–13.9;P ⫽.02). The re-sults remained significant after adjustment for ART (P ⬍ .05). No significant correlation between low CD8⫹ T-lymphocytes and low birth weight was ob-served in this cross-sectional study.

Risk Factors for PE Before Onset of Encephalopathy

For this purpose, we compared the PE-positive group with the AIDS/PE-negative group (32 verti-cally HIV-1-infected children who developed clinical category C during their lives and without neurologic manifestations) in a cross-sectional study within 12 months before PE diagnosis for the PE-positive group (mean time: 8.2 ⫾ 0.4 months) or to clinical category C or AIDS (including lymphoid interstitial pneumonitis [LIP]) for the AIDS/PE-negative group (mean time, 8.5 ⫾1.4 months) (Table 2). Again, the %CD8⫹ T-lymphocytes were significantly lower in the PE-positive group (P ⬍ .01) than in the AIDS/ PE-negative group. Higher proportions of children in the PE-positive group (P ⬍.01) and in the early-onset PE (P⬍.01) and late-onset PE (P⬍.05) groups had CD8⫹ ⬍25% T-lymphocytes before PE than the AIDS/PE-negative group before developing AIDS.

Plasma VL levels were higher in the PE-positive group (P ⫽ .05; Table 2) and in the late-onset PE group (P⬍.05) than in the AIDS/PE-negative group, respectively. Normalized CD8⫹ T-lymphocytes per-centages with respect to seroreverting children were similarly significant (Table 2). Normalized absolute counts of CD8⫹ T-lymphocytes were significantly lower in the 3 groups of children with encephalopa-thy with respect to the AIDS/PE-negative group (data not shown). The biological characteristics of viral isolates more frequently observed in peripheral blood before PE onset were SI phenotype (65%) and rapid/high replication kinetics (65%), both corre-sponding to X4 or lymphocytotropic viral isolates. With respect to AIDS-related events, PE was strongly associated with anemia (P⫽.02) and LIP (P⬍.01) in the PE-positive group (Table 2).

Studies Before and After PE

For each child, immunologic and virologic mark-ers were analyzed in parallel closely before clinical onset of PE (within a 6-month interval before PE; mean time: 4.0 ⫾ 0.6 months) and closely after PE onset (mean time: 3.5 ⫾0.6 months) and compared by using the Student ttest for paired samples. The percentages of both CD4⫹and CD8⫹T-lymphocytes were significantly higher before PE onset with re-spect to the post-PE study (P ⬍ .05; Fig 2A). When analyzing the influence of ART administered after PE emergence on these parameters by analysis of variance for repeated measures of CD4⫹ and CD8⫹ T-lymphocytes and VL adjusting for ART post-PE, only the variation of CD4⫹ T-lymphocytes was sig-nificantly influenced, being the fall-off of CD4⫹ T-lymphocytes more marked in the children not treated (P ⫽.01; Fig 2B).

Peripheral blood monocytes by flow cytometry were similarly analyzed before and after clinical PE onset in the same time points and compared as paired samples. Both percentages and absolute monocyte counts were significantly higher in the

Fig 1. Kaplan-Meier curves of progression to encephalopathy, classified according to birth weight of 2500 g (A) and 25% of CD8⫹

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pre-PE interval than in the post-PE (P⬍.05 andP

.001, respectively) and than in a control group of vertically HIV-1-infected children without neuro-logic manifestations (11 children in clinical category A, 11 in B, and 8 in C; P ⬍ .05 and P ⬍ .001, respectively; Table 3). The differences in monocyte absolute counts between the study pre-PE and the

control group remain significant after rating the con-trol group in clinical categories A, B, and C (P

.001). Besides, absolute counts of monocytes after clinical onset of PE were higher than those of the control group (P⬍ .001; Table 3). Of relevance, in 4 children of our cohort who underwent bone marrow biopsies, extensive histiocytic infiltration with

in-TABLE 2. Clinical and Immunologic Characteristics of the AIDS/PE-Negative and the PE-Posi-tive Groups at a Time Point Before Developing AIDS and PE, RespecPE-Posi-tively

Characteristic AIDS/PE-Negative Group

(n⫽32)

PE-Positive Group (n⫽58)

Age (y) 3.4⫾0.4 2.7⫾0.5

Gender (M/F) 15/17 22/36

CDC clinical and immunologic classification

A 15 (47%) 21 (36%)

B 12 (38%) 32 (55%)

C 5 (15%) 5 (9%)

CD4⫹25% 11 (34%) 25 (38%)

CD4⫹15–25% 8 (25%) 13 (21%)

CD4⫹15% 13 (41%) 18 (41%)

Immunologic and virologic characteristics

%CD4⫹T-lymphocytes 21.42.33 19.32.7

Z%CD4⫹T-lymphocytes 8.40.8 11.41.3

%CD8⫹T-lymphocytes 38.82.5 23.42.7†

Z%CD8⫹T-lymphocytes 11.11.4 4.81.8†

VL (copies/mL) 82 425⫾25 039 204 433⫾69 567*

Log10VL 4.47⫾0.21 5.10⫾0.13*

CD8⫹25% T-lymphocytes 18% 58%†

VL⬎4.5 log10 56% 76%

VL⬎5.5 log10 0% 18%

Non-SI viral phenotype 3 (25%) 7 (35%)

SI viral phenotype 9 (75%) 13 (65%)

Slow/low replication kinetics 3 (25%) 7 (35%)

Rapid/high replication kinetics 9 (75%) 13 (65%)

Clinical characteristics

Hepatomegaly 8 (50%) 28 (48%)

Lymphadenopathy 5 (31%) 23 (40%)

Candidiasis 3 (19%) 22 (38%)

LIP 1 (6%) 17 (29.3%)†

Anemia 2 (13%) 23 (40%)*

Failure to thrive 3 (19%) 13 (22.4%)

HIV-related myocardiopathy 1 (6%) 12 (20.6%)

Trombocytopenia 2 (12.5%) 9 (15.5%)

CDC indicates Centers for Disease Control and Prevention. Results are expressed as mean⫾SEM.

*P⬍.05. †P⬍.01.

Fig 2. A, Comparison of immunologic (%CD4⫹T-lymphocytes and %CD8T-lymphocytes) and virologic variables (log

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creased expression of phagocyte lineage cells to-gether with altered erythropoiesis was found, which would also account for anemia.

Comparison of Early- and Late-Occurring PE in Children

Early-onset PE was found to be more frequently an isolated symptom of AIDS (91%) than late onset PE (24%). Head circumferences of the 37 children with early-onset PE were slightly lower than the 21 with late-onset PE (31.0 vs 32.9 cm, respectively). Simi-larly, the number of premature children was higher in the early-onset PE group (P ⫽ .09), and birth weight was significantly lower in the early-onset PE group than in the late-onset PE group (P ⫽ .02). Cerebral atrophy was the main observed finding by CT scan (n⫽47), regardless of age of PE onset. Basal ganglia calcifications were found in 46% of children who underwent CT, more predominantly in early-onset PE than in late-early-onset PE (69% vs 23%).

DISCUSSION

HIV-1 infection of the CNS remains an important clinical concern. One of the primary challenges in the care of infants and children who are infected with HIV-1 remains the early and accurate diagnosis of PE to initiate early, aggressive, specific ART in hopes of preventing devastating effects of HIV on the devel-oping brain.22The first step toward PE prevention in HIV-1-infected children should be directed at pre-dicting risk of PE and thus the prompt and reliable identification of infants who are at risk for CNS disease progression. Although PE unquestionably shortens life expectancy,3a 4.4-year median survival of our series of 58 HIV-1-infected children with PE must be interpreted in the context of an HIV-1-in-fected population, so we compared background mor-tality in a similar group of children without PE. Future progress should be measured not merely in terms of extending the life expectancy of children with PE but by preventing neurologic damage when it is still possible and therefore in the first stages of CNS infection and particularly toward primary pre-vention of disease.

Whether an HIV-1-infected child develops neuro-logic disease and how early the clinical signs of infection appear are most likely the net result of both viral virulence and host factors. Important viral fac-tors include cell tropism and strains that determine neurovirulence. The host factors include the cellular expression of viral co-receptors and maintenance of competent immune responses. The conclusions drawn by different cross-sectional and longitudinal

studies in HIV-1-infected children to date have failed to estimate the timing of onset of PE with the routine progression markers and other parameters used in the follow-up of HIV-1-infected children in clinical practice. Thus, the longitudinal nature of this study enables some inferences to be drawn concerning the timing of the onset of neurologic abnormalities. In that context, CD8⫹T-lymphocyte counts and mono-cytes may serve to predict the risk of PE and the time of clinical emergence, respectively.23

Evidence for higher peripheral monocytes in adult patients with HIV-1-associated dementia than in pa-tients without dementia, with a unique subset of activated monocytes, once encephalopathy is estab-lished, has been previously reported.24,25 More im-portant, a peak of significantly higher peripheral monocytes before the onset of PE with respect to established PE has not been previously described, and strengthens the growing evidence that an in-creased traffic of monocytes to the brain may be a key factor in triggering neurologic symptoms. Mono-nuclear phagocytes (brain macrophages and micro-glia) are the main HIV-1 reservoir in the CNS, and the abundance of macrophages in the brain seems to be the best correlate with severity of PE.26 –28 How-ever, the peak of monocytes may also indicate a reaction to PE in its onset, and not a pathogenic mechanism.

The higher frequency of PE as AIDS-defining ill-ness when occurring before 2 years of age has been previously described,1,3,4 suggesting that the key pathogenic mechanisms responsible for progression of PE may vary with age of onset. In this regard, it has been suggested that HIV-1-infected infants with rapid progression are reflecting in utero HIV-1 infec-tion.16,29 –31Likewise, early-onset PE may be the con-sequence of in utero infection of the brain.1 Head circumference, in utero exposure to illicit drugs, and birth weight were prognostic markers for progres-sion to PE.1However, in both early- and late-onset PE, the entry of the virus into the CNS takes place during the development of the brain and during the process of myelination. HIV-1 encephalopathy in young children could be a very specific consequence of the interaction between HIV-1 and the developing immune and nervous systems and thus might re-quire a specific therapeutic approach.1

We previously demonstrated in a prospective study that CD8⫹ T-lymphocyte counts in the first months of life were significantly associated with the risk for emergence of neurologic signs defining prob-able PE (cognitive deterioration, cerebral atrophy, progressive motor dysfunction, gait disorder, reflex

TABLE 3. Monocyte Blood Counts by Flow Cytometry in HIV-Infected Children

% Monocytes Monocytes/␮L

Controls HIV-1 P* Controls HIV-1 P*

Pre-PE 7.8⫾1.5 11.3⫾1.6 .03 180.1⫾27.2 794.3⫾76.1 ⬍.001

Post-PE 7.77⫾0.8 .25 567.2⫾85.5 ⬍.001

P .03 ⬍.001

Comparison of levels before and after the onset of PE with a control group of children without PE in the 3 CDC clinical categories.

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disturbances, abnormal muscle tone, and HIV-1 en-cephalitis.15 Of note, there were not differences in confounding factors, particularly equal number of patients exposed to drugs in utero. The most striking result is that among all of the immunologic and virologic variables assessed in this observational study, the only significant difference during the first months of life and before the onset of PE are the CD8⫹ T-lymphocytes.

Cytotoxic CD8⫹ T-lymphocytes (CTLs) kill virus-infected cells, and their role in the control of HIV-1 infection in vitro and in vivo is well established, although only a few studies have analyzed their prognostic value in the follow-up of infected adults32–34 and children.21,35,36 The suppression of HIV-1 replication is dependent on the presence of a relatively small number of HIV-1-specific CD8⫹ T-lymphocyte clones, and it is possible that the dura-tion of the neurologically asymptomatic phase for any given child may depend mostly on the magni-tude of specific CD8⫹ T-lymphocyte responses.37 Thus, a decrease of CD8⫹T-lymphocytes would di-minish the host capacity to control viral infection, as reported in animal models,38enabling infected mac-rophages to cross the blood-brain barrier. Compati-ble with this, 1-infected macrophages by HIV-1-specific CTLs have been reported to be less effectively destroyed than HIV-1-infected CD4⫹ T-lymphocytes.39Therefore, a drop in number of CTLs will favor escape of circulating infected macrophages from lysis. Recent studies have shown increased numbers of activated CD8⫹ T-lymphocytes in the brain correlating with neurologic dysfunction in an-imal models40 or in specimen brain biopsies from HIV-infected children with encephalopathy.41

Although some authors have advocated the key significance of high VL for the increased traffic of peripheral monocytes into brain in adults HIV-1-associated dementia,25 we did not find significant differences between children with and without PE, in accordance with recent results in adult patients.42,43 In fact, children show extremely high VL levels since early in life,44 – 47even under highly active ART, rul-ing out its possible role as a triggerrul-ing factor for the onset of the neurologic picture. There was a trend for SI viral phenotype with rapid/high replication kinet-ics before PE emergence, as has been described soon before progression to AIDS in children.48 However, given the compartmentalization of HIV, circulating viral isolates may only be reflecting the progression of the disease but not specifically indicating the course of CNS involvement.49 Anemia was a good predictor of PE in agreement with other authors3,5 and may suggest in part hematopoiesis impair-ment.25,50Also, we observed LIP before PE in a sig-nificantly higher number of children than in an age-matched group of HIV-positive children before undergoing non-PE AIDS-defining illnesses.

Our results advocate the use of CD8⫹ T-lympho-cyte and monoT-lympho-cyte counts to follow-up HIV-1-in-fected children. We suggest that CD8⫹ T-lympho-cytes may be the nexus for many different aspects of the disease, namely loss of control of HIV-1 replica-tion determining higher VL, increased traffic of

acti-vated and/or infected monocytes, spread of infection to immune sanctuaries, and finally clinical neuro-logic emergence of PE. Moreover, we suggest that CD8⫹ T-lymphocytes and/or monocytes may be used as putative biological markers of neuropatho-genicity. This might suggest their use in decision making of when to start more effective ART regi-mens for HIV-1 infection of the CNS and the need of new therapies either to preserve or to augment an adequate CTL immune response. Early detection of children who are at risk for developing PE is partic-ularly important because aggressive highly active antiretroviral therapy improves neurologic symp-toms, allows possible use of neuroprotective treat-ment to prevent further developtreat-ment of encephalop-athy, and emphasizes the relevance of developing therapies aimed to enhance CD8⫹ T-lymphocyte function.51

CONCLUSION

The surrogate markers routinely used in clinical practice for HIV-1 infection (CD4⫹ T-lymphocyte counts and VL) seem to be insufficient to evaluate the clinical involvement of the CNS. Other systemic markers, as the recent proposed markers for PE evo-lution (CSF VL by lumbar puncture52 and brain at-rophy by cerebral magnetic resonance imaging38) are undoubtedly more invasive than measuring CD8⫹ T-lymphocyte and monocyte counts, when the neu-rologic manifestations of PE are still preventable.

ACKNOWLEDGMENTS

This work was funded by grants of the Fundacio´n para la Investigacio´n y la Prevencio´n del SIDA en Espan˜a (FIPSE 3008/ 99), Fondo de Investigacio´n Sanitaria (00/0207), Programa Nacio-nal de Salud (SAF 99 – 0022), and the Comunidad de Madrid.

We thank Dolores Garcı´a Alonso for excellent technical assis-tance.

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DOI: 10.1542/peds.111.2.e168

2003;111;e168

Pediatrics

Dolores Gurbindo, José-Tomás Ramos and M?Angeles Muñoz-Fernández

Silvia Sánchez-Ramón, José M? Bellón, Salvador Resino, Carmen Cantó-Nogués,

Predictors of HIV-1-Associated Progressive Encephalopathy in Children

T-Lymphocytes and High Circulating Monocytes Are

+

Low Blood CD8

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DOI: 10.1542/peds.111.2.e168

2003;111;e168

Pediatrics

Dolores Gurbindo, José-Tomás Ramos and M?Angeles Muñoz-Fernández

Silvia Sánchez-Ramón, José M? Bellón, Salvador Resino, Carmen Cantó-Nogués,

Predictors of HIV-1-Associated Progressive Encephalopathy in Children

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Figure

TABLE 1.Clinical and Immunologic Characteristics of the PE-Negative and the PE-PositiveGroups at Birth and at a Time Point Within the First Months of Life (Range: 2–7 Months)
Fig 1. Kaplan-Meier curves of progression to encephalopathy, classified according to birth weight of 2500 g (A) and 25% of CD8�T-lymphocytes (B).
TABLE 2.Clinical and Immunologic Characteristics of the AIDS/PE-Negative and the PE-Posi-tive Groups at a Time Point Before Developing AIDS and PE, Respectively

References

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